Methylene Blue

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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To view the Notice from the Expert Committee that posted in conjunction with this accelerated revision, please click https://www.uspnf.com/rb-methylene-blue-20230929.

C₁₆H₁₈ClN₃S · xH₂O

Phenothiazin-5-ium, 3,7-bis(dimethylamino)-, chloride, hydrate (1:1:x);

3,7-Bis(dimethylamino)phenothiazin-5-ium chloride hydrate; CAS RN^®: 122965-43-9.

Pentahydrate 409.93 CAS RN^®: 32680-41-4.

Trihydrate 373.90 CAS RN^®: 7220-79-3; UNII: T42P99266K.

Monohydrate 337.90 CAS RN^®: 67183-68-0.

Anhydrous 319.85 CAS RN^®: 61-73-4; UNII: 8NAP7826UB.

1 DEFINITION

Methylene Blue contains NLT 97.0% and NMT 103.0% of methylene blue (C₁₆H₁₈ClN₃S), calculated on the dried basis.

2 IDENTIFICATION

A. SPECTROSCOPIC IDENTIFICATION TESTS 〈197〉, Infrared Spectroscopy: 197A or 197K

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

C. IDENTIFICATION TESTS-GENERAL 〈191〉, Chemical Identification Tests, Chloride

Sample solution: Ignite 50 mg of Methylene Blue with 0.5 g of anhydrous sodium carbonate. Cool and dissolve the residue in 10 mL of 2 N nitric acid. Filter and use 2 mL of the filtrate for performing the test.

Acceptance criteria: Meets the requirements

3 ASSAY

3.1 PROCEDURE

All solutions containing methylene blue should be prepared fresh before analysis.

Solution A: 0.1% trifluoroacetic acid in water

Solution B: Acetonitrile

Diluent: Solution A and Solution B (70:30)

Mobile phase: See Table 1.

Table 1

Standard solution: 1 mg/mL of USP Methylene Blue RS in Diluent. Stirring and sonication may be necessary for complete dissolution.

Sample solution: 1 mg/mL of Methylene Blue in Diluent. Stirring and sonication may be necessary for complete dissolution.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 246 nm

Column: 4.6-mm × 10-cm; 3.5-µm packing L11

Column temperature: 30°

Flow rate: 1 mL/min

Injection volume: 5 µL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 3.0

Relative standard deviation: NMT 1.10%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of methylene blue (C₁₆H₁₈ClN₃S) in the portion of Methylene Blue taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of methylene blue from the Sample solution

r_S = peak response of methylene blue from the Standard solution

C_S = concentration of USP Methylene Blue RS in the Standard solution (mg/mL)

C_U = concentration of Methylene Blue in the Sample solution (mg/mL)

Acceptance criteria: 97.0%–103.0% on the dried basis

4 IMPURITIES

RESIDUE ON IGNITION 〈281〉: NMT 0.15%

ORGANIC IMPURITIES

Diluent, Mobile phase, Standard solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

System suitability solution: 1 mg/mL of USP Methylene Blue RS and 0.025 mg/mL of USP Azure B RS in Diluent

Sensitivity solution: 0.5 µg/mL of USP Methylene Blue RS in Diluent from the Standard solution

System suitability

Samples: System suitability solution and Sensitivity solution

[Note—The relative retention times for azure B and methylene blue are 0.8 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 3.5 between methylene blue and azure B peaks, System suitability solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis

Sample: Sample solution

Calculate the percentage of azure B or any unspecified impurity in the portion of Methylene Blue taken:

Result = (r_U/r_T) × 100

r_U = peak response of azure B or any unspecified impurity

r_T = sum of all the peak responses

Acceptance criteria: See Table 2. The reporting threshold is 0.05%.

Table 2

^a 3-(Dimethylamino)-7-(methylamino)phenothiazin-5-ium chloride.

^b Total impurities does not include azure B.

5 SPECIFIC TESTS

Change to read:

LOSS ON DRYING 〈731〉

Analysis: Dry at 105° for 5 h.

Acceptance criteria: 8.0%–24.0% (RB 1-Sep-2023)

BACTERIAL ENDOTOXINS TEST 〈85〉: The level of bacterial endotoxins is such that the requirement under the relevant dosage form monograph(s) in which Methylene Blue is used can be met. Where the label states Methylene Blue must be subjected to further processing during the preparation of injectable dosage forms, the level of bacterial endotoxins is such that the requirement under the relevant dosage form monograph(s) in which Methylene Blue is used can be met.

MICROBIAL ENUMERATION TESTS 〈61〉 and TESTS FOR SPECIFIED MICROORGANISMS 〈62〉: The total aerobic microbial count is NMT 10² cfu/g, and the total combined yeasts and molds is NMT 10¹ cfu/g.

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in well-closed containers and protect from light. Store below 30°.

LABELING: Where Methylene Blue must be subjected to further processing during the preparation of injectable dosage forms to ensure acceptable levels of bacterial endotoxins, it is so labeled.

USP REFERENCE STANDARDS 〈11〉

USP Azure B RS

3-(Dimethylamino)-7-(methylamino)phenothiazin-5-ium chloride.

C₁₅H₁₆ClN₃S  305.82

USP Methylene Blue RS